1. Field of the Invention
The present invention relates to the use of α-unsubstituted β-amino acids and especially derivatives thereof as chiral selectors for separating substance mixtures, preferably mixtures of chiral substances, more preferably enantiomer mixtures, especially enantiomers of substances selected from the group comprising β-amino acids and derivatives thereof, α-amino acids and α-hydroxy acids, and to chiral stationary phases (CSPs) which comprise α-unsubstituted β-amino acids and especially derivatives thereof as central chiral selectors, to their synthesis and to processes for separating substance mixtures, preferably mixtures of chiral substances, more preferably enantiomer mixtures, especially enantiomers of substances selected from the group comprising β-amino acids and derivatives thereof, α-amino acids and α-hydroxy acids with the aid of these stationary phases.
2. Discussion of the Background
The rising demand for enantiomerically pure substances and active ingredients for chemical and pharmaceutical applications has led to the development of a multitude of stereoselective separation technologies, in particular in the field of chromatography, which can be used either on the analytical scale for controlling enantiomeric purity and checking racemization processes, for pharmaceutical quality control and for pharmacokinetic studies, or on the preparative scale for providing enantiomerically pure compounds.
In contrast to diastereomers, enantiomers have identical chemical and physical properties in an achiral environment. Chromatographic enantiomer separations can therefore be carried out either using indirect methods, i.e. after reacting the analyte with a chiral derivatizing reagent to give a diastereomer mixture which, unlike an enantiomer mixture, can be separated on achiral phase material, or with the aid of so-called direct methods using a chiral selector which is incorporated into the mobile or stationary phase. The separating performance is based here on the different stability of the diastereomeric complexes which are formed by noncovalent interactions between the analyte and the selector.
Direct and also indirect processes have already found use in the field of enantiomer separation in various chromatographic and electrophoretic methods such as gas chromatography (GC), high-performance liquid chromatography (also high-pressure liquid chromatography, HPLC), thin-layer chromatography (TLC), super- and subcritical liquid chromatography (SLC), capillary electrochromatography (CEC) and capillary electrophoresis (CE) (Gübitz and Schmid (Eds.), Methods in Molecular Biology, Vol. 243: Chiral Separations: Methods and Protocols, Humana Press Inc: Totowa, N.J.: 2004; Gübitz und Schmid, Biopharm. Drug Dispos. 2001, 22, 291-336).
A derivatization of the analyte means at least one additional reaction step, which can lead to the formation of undesired by-products and decomposition products and to (partial) racemization. In addition, suitable functional groups must be present in the analyte for derivatization, and the chiral derivatizing reagent must be available in high enantiomeric purity (Gübitz and Schmid, Biopharm. Drug Dispos. 2001, 22, 291-336), which is why nonderivative direct chromatographic or electrophoretic methods are nowadays preferred. Although the addition of a chiral selector to the mobile phase of a chromatographic or electrophoretic system is a simple method for enantiomer separation in terms of handling, it is very expensive and not practicable in all cases.
Direct chromatographic processes using chiral stationary phases in which one chiral selector is bonded covalently or adsorptively to a support material are convenient in terms of handling and—assuming sufficient separating performance of the chiral phase material—also employable on the preparative scale. Specifically in the field of chiral stationary phases, a chiral selector which, on the one hand, allows an efficient separation of the two enantiomers of a chiral compound, but, on the other hand, is flexible enough to allow application for a wide class of compounds is desirable.
One variant of the direct methods is that of ligand exchange processes (LE), which are based on the formation of ternary mixed complexes between a metal ion, a chiral selector and the analyte, both of which function as ligands on the metal ion. What are responsible here for a successful separation are the different stability constants of the mixed complexes with the (R) and (S) enantiomers of the analyte. The ligand exchange principle has been used successfully for enantiomer separation in a number of the processes mentioned above: firstly with addition of a chiral selector to the electrolyte in capillary electrophoresis and secondly using chiral stationary phases in classical column chromatography, high-performance liquid chromatography, thin-layer chromatography and capillary electrochromatography, in which cases the chiral selector may be bonded covalently or adsorptively to the support material. Since, however, chiral separations cannot be forecast to the present day, it is still a great chromatographic challenge to find the suitable combination of chiral stationary and mobile phase efficiently and rapidly (Subramanian, Practical Approach to Chiral Separations by Liquid Chromatography, Wiley-VCH: Weinheim 1994; Gübitz and Schmid (Eds.), Methods in Molecular Biology, Vol 243: Chiral Separations: Methods and Protocols, Humana Press Inc: Totowa, N.J.: 2004; Francotte, GIT Labor-Fachzeitschrift 5/2006, 452-455), and intensive studies have led to always novel and improved phase materials (Lämmerhofer and Lindner, In: Separation Methods in Drug Synthesis and Purification, Valkó (Ed.), Elsevier: Amsterdam 2000, 337-437). According to Armstrong et al. (Anal. Chem. 2001, 73, 557A-561A), as early as 2001, more than 100 chiral stationary phases based on different selectors were commercially available for high-performance liquid chromatography alone. Every manufacturer offers comprehensive application handbooks, in which the wide variety of different separating conditions, for example for aliphatic, aromatic, alicyclic or heterocyclic chiral amines, alcohols, amino alcohols or α-amino acids and derivatives thereof, are listed almost exclusively in a product-specific manner. This multitude of commercially available chiral stationary phases firstly demonstrates the immense significance and the great interest in these separation techniques, but also makes clear a great disadvantage which afflicts direct chromatographic and electrophoretic methods with the aid of chiral stationary phases to the present day: often, a series of (expensive) chiral stationary phases is needed to achieve an efficient separation even in the case of structurally closely related units.
Thus, in-house studies and also literature data demonstrate that, to date, a multitude of stationary phases which are then used in gas chromatography (GC) or liquid chromatography (for example HPLC) are needed for chiral chromatography, for example of β-amino acids and derivatives thereof. Conventionally known phases include, for example, cellulose carbamate phases, amylose derivatives, crown ethers (Berkecz et al., J. Chromatogr. A, 2006, 1125, 138-143; Hyun et al., J. Sep. Sci. 2005, 28, 421-427), ligand exchange phases (Hyun et al., J. Sep. Sci. 2003, 26, 1615-1622; Hyun et al., Biomed. Chromatogr. 2003, 17, 292-296) or macrocyclic glycopeptide phases (Sztojkov-Ivanov et al., Chromatographia 2006, 64, 89-94; Illisz et al., J. Sep. Sci. 2006, 29, 1305-1321 and literature cited there; D'Acquarica et al., Tetrahedron: Asymmetry 2000, 11, 2375-2385).
In the last few years, owing to their exceptional pharmacological properties, β-amino acids have been incorporated as key components into a multitude of peptidomimetics and further biologically active substances (Kuhl et al., Amino Acids 2005, 29, 89-100). Associated with this has also been the development of a rising demand for analytical methods for testing the enantiomeric purity of the synthesis units and end products, specifically also in the sector of minor trace determination for detection of traces of one enantiomer in the presence of a significant excess of the optical antipode (Juaristi and Soloshonok, Enantioselective Synthesis of β-Amino Acids, Wiley-VCH: New York 2005). It is an object of the present invention to provide novel chiral selectors, on the basis of which novel chiral phases for separating substance mixtures, preferably mixtures of chiral substances, more preferably enantiomer mixtures, especially enantiomers of substances selected from the group comprising β-amino acids and derivatives thereof, α-amino acids and α-hydroxy acids, can be provided, which allow efficient and very substantially universal separation of enantiomer pairs of chiral compounds, especially of β-amino acids and derivatives thereof, with chromatographic methods, especially with the aid of high-performance liquid chromatography, on the analytical and preparative scale.
It has now been found that, completely surprisingly, α-unsubstituted β-amino acid derivatives are flexible and nevertheless highly selective chiral selectors in processes for separating substance mixtures, preferably mixtures of chiral substances, more preferably enantiomer mixtures, especially β-amino acids and derivatives thereof. Further substance classes for which the selectors of the invention can be used for enantiomer separation are, for example, α-amino acid and α-hydroxy acids. It is thus a particular advantage of the present invention that the chiral phases which comprise chiral selectors based on α-unsubstituted β-amino acid derivatives are surprisingly usable universally for chromatographic separation of a large number of substance classes.